The evolutionary conserved proteins CEP90, FOPNL, and OFD1 recruit centriolar distal appendage proteins to initiate their assembly

Abstract

In metazoa, cilia assembly is a cellular process that starts with centriole to basal body maturation, migration to the cell surface, and docking to the plasma membrane. Basal body docking involves the interaction of both the distal end of the basal body and the transition fibers/distal appendages, with the plasma membrane. Mutations in numerous genes involved in basal body docking and transition zone assembly are associated with the most severe ciliopathies, highlighting the importance of these events in cilium biogenesis. In this context, the ciliate Paramecium has been widely used as a model system to study basal body and cilia assembly. However, despite the evolutionary conservation of cilia assembly events across phyla, whether the same molecular players are functionally conserved, is not fully known. Here, we demonstrated that CEP90, FOPNL, and OFD1 are evolutionary conserved proteins crucial for ciliogenesis. Using ultrastructure expansion microscopy, we unveiled that these proteins localize at the distal end of both centrioles/basal bodies in Paramecium and mammalian cells. Moreover, we found that these proteins are recruited early during centriole duplication on the external surface of the procentriole. Functional analysis performed both in Paramecium and mammalian cells demonstrate the requirement of these proteins for distal appendage assembly and basal body docking. Finally, we show that mammalian centrioles require another component, Moonraker (MNR), to recruit OFD1, FOPNL, and CEP90, which will then recruit the distal appendage proteins CEP83, CEP89, and CEP164. Altogether, we propose that this OFD1, FOPNL, and CEP90 functional module is required to determine in mammalian cells the future position of distal appendage proteins.

Human SFI1 and Centrin form a complex critical for centriole architecture and ciliogenesis

“…The EMBO Journal 41: e112107 | 2022 been recently proposed to be at the base of appendages (Kumar et al, 2021;Le Borgne et al, 2022). We did not observe any major defect of CEP90 localization (Fig 6E-H, J and K), suggesting that CEP164 defects may be due to an as-yet undescribed direct regulatory mechanism between the SFI1 protein and CEP164.…”

“…We next sought an alternative explanation and tested whether appendage anchoring on triplet microtubules might be affected. To do so, we stained for the protein CEP90, which has been recently proposed to be at the base of appendages (Kumar et al , 2021; Le Borgne et al , 2022). We did not observe any major defect of CEP90 localization (Fig 6E–H, J and K), suggesting that CEP164 defects may be due to an as‐yet undescribed direct regulatory mechanism between the SFI1 protein and CEP164.…”

“…Length, diameter, protein coverage, and relative protein position quantifications were performed as previously published in (Le Guennec et al , 2020). To generate the panels in Figs 1D, F, H and J and 3D–F, we used two homemade plugins for ImageJ as described previously (Le Borgne et al , 2022).…”

Single-molecule localization microscopy reveals the ultrastructural constitution of distal appendages in expanded mammalian centrioles

“…Moreover, CEP83 was previously reported to own a minimum measure among the outer DAP structure 28 . Recent studies further indicate that MNR, FOPNL, CEP90, and OFD1 are found near the DAP base between CEP83 and the microtubule triplet 21 , 22 . Nevertheless, their ultrastructural arrangement at a molecular level remains to be investigated.…”

“…Numerous DAP proteins have been identified in mammalian cells for their roles in ciliogenesis and ciliary-associated regulations 10,[15][16][17][18][19] . To initiate DAP assembly, C2CD3 is recruited to the centriole-distal end 18,20 and MNR, FOPNL, CEP90, and OFD1 are indispensable for further recruitment of CEP83, CEP89, and SCLT1 21,22 . SCLT1 is then required for FBF1 and CEP164 recruitment 10 .…”